molecules

Article Fluorinated Analog NMR s of from (Allium sativum): Synthesis, and Anti-Angiogenesis and Antithrombotic Studies

Eric Block 1,* ID , Benjamin Bechand 1, Sivaji Gundala 1, Abith Vattekkatte 1, Kai Wang 1, Shaymaa S. Mousa 2, Kavitha Godugu 2, Murat Yalcin 2,3 and Shaker A. Mousa 2,* ID

1 Department of Chemistry, University at Albany, State University of New York, Albany, NY 12222, USA; [email protected] (B.B.); [email protected] (S.G.); [email protected] (A.V.); [email protected] (K.W.) 2 The Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, Rensselaer, NY 12144, USA; [email protected] (S.S.M.); [email protected] (K.G.); [email protected] (M.Y.) 3 Department of Physiology, Veterinary Medicine Faculty, Uludag University, Bursa 16059, Turkey * Correspondence: [email protected] (E.B.); [email protected] (S.A.M.); Tel.: +1-518-442-4459 (E.B.); +1-518-694-7397 (S.A.M.)

Received: 6 October 2017; Accepted: 24 November 2017; Published: 28 November 2017

Abstract: We describe the synthesis, reactivity, and antithrombotic and anti-angiogenesis activity of difluoroallicin (S-(2-fluoroallyl) 2-fluoroprop-2-ene-1-sulfinothioate) and S-2-fluoro-2-propenyl-L- cysteine, both easily prepared from commercially available 3-chloro-2-fluoroprop-1-ene, as well as the synthesis of 1,2-bis(2-fluoroallyl)disulfane, 5-fluoro-3-(1-fluorovinyl)-3,4-dihydro-1,2-dithiin, trifluoroajoene ((E,Z)-1-(2-fluoro-3-((2-fluoroallyl)sulfinyl)prop-1-en-1-yl)-2-(2-fluoroallyl)disulfane), and a bis(2-fluoroallyl)polysulfane mixture. All tested organosulfur compounds demonstrated effective inhibition of either FGF or VEG-mediated angiogenesis (anti-angiogenesis activity) in the chick chorioallantoic membrane (CAM) or the mouse Matrigel® models. No embryo mortality was observed. Difluoroallicin demonstrated greater inhibition (p < 0.01) versus organosulfur compounds tested. Difluoroallicin demonstrated dose-dependent inhibition of angiogenesis in the mouse Matrigel® model, with maximal inhibition at 0.01 mg/implant. Allicin and difluoroallicin showed an effective antiplatelet effect in suppressing aggregation compared to other organosulfur compounds tested. In platelet/fibrin clotting (anti-coagulant activity), difluoroallicin showed concentration-dependent inhibition of clot strength compared to allicin and the other organosulfur compounds tested.

Keywords: garlic; allicin; vinyl dithiins; ajoene; difluoroallicin; trifluoroajoene; angiogenesis; anti-angiogenesis; platelet; coagulation; thrombosis; anti-thrombotic; 2-fluoroallyl compounds

1. Introduction Since its discovery by Cavallito in 1944, the antibiotic active principle of garlic, allicin (1), has been the subject of extensive research [1–3]. Enzymatically released from its precursor alliin (2) when garlic is crushed, allicin is both unstable and reactive, readily decomposing by way of 2-propenesulfenic acid (3) and thioacrolein (5) to diallyl disulfide (4) and homologous polysulfides (polysulfanes), vinyl 1,2- and 1,3-dithiins (6 and 7, respectively) and ajoene (8) (Scheme1), among other products, all of which have interesting biological properties, e.g., as antiplatelet and anticancer agents [1–8]. Given the great importance of fluorine in medicinal chemistry and chemical biology [9,10], we were interested in

Molecules 2017, 22, 2081; doi:10.3390/molecules22122081 www.mdpi.com/journal/molecules Molecules 2017, 22, 2081 2 of 20

[9,10], we were interested in the effect that fluorine substitution would have on the chemical reactivity and biological activity of these compounds, since such fluorinated analogs are presently unknown. Compounds with C–F bonds, particularly of the sp2-C–F type, offer advantages associated with the small size, high electronegativity, and metabolic stability associated with the fluorine as well as the altered molecular lipophilicity, membrane fluidity, binding affinity, enhanced volatility, and possibilities for halogen bonding [11] and conformational preferences of the fluorinated compounds compared to their analogs. We also follow up on our previous work on trifluoroselenomethionine [12]. group reactivity, a defining feature of the biological activity of allicin and other garlic compounds [13–15], should be enhanced by H/F substitution in garlic thioallyl compounds. Three types of C–F substituted allyl groups were considered for this study, namely CH2=CFCH2– X (A), CF2=CFCH2–X (B), and CF2=CFCF2–X (C). We chose type A based on commercial availability of CH2=CFCH2Cl (3-chloro-2-fluoroprop-1-ene (9) (Scheme 2), as well as on our inability to convert thioacetate CF2=CFCH2SAc (from Mitsunobu reaction of CF2=CFCH2OH [16]) to the corresponding disulfide, and on the relative inaccessibility of precursors C, such as CF2=CFCF2OSO2F [17]. Here, we describe our efforts to synthesize and study the reactivity and activity of difluoroallicin 12 (S-(2- fluoroallyl) 2-fluoroprop-2-ene-1-sulfinothioate), easily prepared from 9, and related compounds such as S-2-fluoro-2-propenyl-L-cysteine (fluorodeoxyalliin, 13), 1,2-bis(2-fluoroallyl)disulfane (11), 5- Molecules 2017, 22, 2081 2 of 20 fluoro-3-(1-fluorovinyl)-3,4-dihydro-1,2-dithiin (16), trifluoroajoene ((E,Z)-1-(2-fluoro-3-((2-fluoro- allyl)sulfinyl)prop-1-en-1-yl)-2-(2-fluoroallyl)disulfane, 18), and 1,2-bis(2-fluoroallyl)polysulfane (21). Wethe effectalso describe that fluorine the anti-angiogenesis substitution would and haveantithro on thembotic chemical activity reactivity of some andof these biological compounds. activity of theseCancer compounds, progression since such is known fluorinated to be analogsaccelerated are presentlyby angiogenesis unknown. and Compounds thrombosis with(platelet/fibrin) C–F bonds, particularlystimulation, ofwhich the sp in2-C–F turn type, are offermediated advantages by vari associatedous factors with secreted the small by size, cancer high cells electronegativity, [18–20]. We previouslyand metabolic showed stability that garlic-derived associated with bioactive the fluorine compounds as well modulate as the altered angiog molecularenesis [5], which lipophilicity, might impactmembrane cancer fluidity, progression binding affinity,and suggests enhanced potent volatility,ial cancer and possibilitieschemoprevention for halogen by organosulfur bonding [11] compounds,and conformational as we further preferences examine of the in fluorinated this study. compounds Additionally, compared studies tosuggest their hydrogenpotential analogs.platelet Wefunction also followmodulation up on by our garlic previous derived work bioactive on trifluoroselenomethionine compounds [21,22]. Our [12 current]. Thiol studies group compared reactivity, thea defining efficacy feature of fluorinated of the biological and non-fluorinated activity of allicin organosulfur and other compounds garlic compounds on angiogenesis [13–15], shouldactivation be byenhanced either bFGF by H/F or VEGF substitution as well in as garlic their thioallylcomparative compounds. efficacy on platelet and coagulation activation.

Scheme 1. EnzymaticEnzymatic formation formation of of allicin allicin ( (11)) from from alliin alliin ( (22)) by by way of 2-propenesulfenic2-propenesulfenic acid acid ( (3),), synthesis of allicin by oxidation of diallyl disulfide disulfide ( 4), conversion of 1 to ajoene ( 8), and decomposition of 1 toto vinyl vinyl dithiins dithiins ( (66 andand 77)) via via intramolecular intramolecular elimination elimination to to thioacrolein thioacrolein (5 ().5 ).

Three types of C–F substituted allyl groups were considered for this study, namely CH2=CFCH2–X (A), CF2=CFCH2–X (B), and CF2=CFCF2–X (C). We chose type A based on commercial availability of CH2=CFCH2Cl (3-chloro-2-fluoroprop-1-ene (9) (Scheme2), as well as on our inability to convert thioacetate CF2=CFCH2SAc (from Mitsunobu reaction of CF2=CFCH2OH [16]) to the corresponding disulfide, and on the relative inaccessibility of precursors C, such as CF2=CFCF2OSO2F[17]. Here, we describe our efforts to synthesize and study the reactivity and activity of difluoroallicin 12 (S-(2-fluoroallyl) 2-fluoroprop-2-ene-1-sulfinothioate), easily prepared from 9, and related compounds such as S-2-fluoro-2-propenyl-L-cysteine (fluorodeoxyalliin, 13), 1,2-bis(2-fluoroallyl)disulfane (11), 5-fluoro-3-(1-fluorovinyl)-3,4-dihydro-1,2-dithiin (16), trifluoroajoene ((E,Z)-1-(2-fluoro-3-((2-fluoroallyl)sulfinyl)prop-1-en-1-yl)-2-(2-fluoroallyl)disulfane, 18), and 1,2-bis(2- fluoroallyl)polysulfane (21). We also describe the anti-angiogenesis and antithrombotic activity of some of these compounds. Cancer progression is known to be accelerated by angiogenesis and thrombosis (platelet/fibrin) stimulation, which in turn are mediated by various factors secreted by cancer cells [18–20]. We previously showed that garlic-derived bioactive compounds modulate angiogenesis [5], which might impact cancer progression and suggests potential cancer chemoprevention by organosulfur compounds, as we further examine in this study. Additionally, studies suggest potential platelet function modulation by garlic derived bioactive compounds [21,22]. Our current studies compared the efficacy of fluorinated and non-fluorinated organosulfur compounds on angiogenesis activation by either bFGF or VEGF as well as their comparative efficacy on platelet and coagulation activation. Molecules 2017, 22, 2081 3 of 20 Molecules 2017, 22, 2081 3 of 20

SchemeScheme 2. Synthesis 2. Synthesis of of difluoroallicin difluoroallicin (12)) from from 1-chloro-2-fluoro-2- 1-chloro-2-fluoro-2-propene (9) by ( 9way) by of way thioacetate of thioacetate 10 10 andand bis(2-fluoro-2-propenyl) bis(2-fluoro-2-propenyl) disulfide disulfide (11), (synthesis11), synthesis of S-2-fluoro-2-propenyl- of S-2-fluoro-2-propenyl-L-cysteine (L13-cysteine) from 9, (13) fromconversion9, conversion of 12 ofto vinyl12 to dithiins vinyl dithiins16 and 1716 viaand 2-fluorothioacrolein17 via 2-fluorothioacrolein (15) with loss (of15 2-fluoro-2-) with loss of propenesulfenic acid (14), conversion of 12 to trifluoroajoene 18, Diels-Alder trapping of 15 with ethyl 2-fluoro-2-propenesulfenic acid (14), conversion of 12 to trifluoroajoene 18, Diels-Alder trapping acrylate giving thiopyrans 19 and 20, conversion of 11 to polysulfane mixture 21 and conversion of 13 of 15 with ethyl acrylate giving thiopyrans 19 and 20, conversion of 11 to polysulfane mixture 21 and to (+)-S-2-fluoro-2-propenyl-L-cysteine S-oxide (22). conversion of 13 to (+)-S-2-fluoro-2-propenyl-L-cysteine S-oxide (22). 2. Results and Discussion 2. Results and Discussion 2.1. Synthesis of Fluorinated Analogues of Garlic-Derived Organosulfur Compounds 2.1. Synthesis of Fluorinated Analogues of Garlic-Derived Organosulfur Compounds It was anticipated that difluoroallicin 12 could be prepared by oxidation of 1,2-bis(2- Itfluoroallyl)disulfane was anticipated (11 that), which difluoroallicin in turn could be12 synthesizedcould be from prepared commercially by oxidation available 1-chloro- of 1,2-bis(2- fluoroallyl)disulfane2-fluoro-2-propene ((119).), Thus, which 9 was in stirred turn overnight could be with synthesized potassium thioacetate from commercially in THF giving S available-(2- 1-chloro-2-fluoro-2-propenefluoroallyl) ethanethioate ( (109).) in Thus, 44% yield.9 was Hydrolysis stirred overnight of 10 (NaOMe/MeOH) with potassium followed thioacetate by treatment in THF with iodine gave 11. Treatment of a chloroform solution of 11 with peracetic acid gave difluoroallicin giving S-(2-fluoroallyl) ethanethioate (10) in 44% yield. Hydrolysis of 10 (NaOMe/MeOH) followed 12 (S-(2-fluoroallyl) 2-fluoroprop-2-ene-1-sulfinothioate) in 42% yield as a yellow liquid with an by treatment with iodine gave 11. Treatment of a chloroform solution of 11 with peracetic acid gave -like odor. When a chloroform solution of 12 was heated in a sealed tube at 80 °C for 4 h it difluoroallicindecomposed12 giving(S-(2-fluoroallyl) a mixture of 2-fluoroprop-2-ene-1-sulfinothioate) 5-fluoro-3-(1-fluorovinyl)-3,4-dihydro-1,2-dithiin in 42% yield (16) as as a the yellow major liquid ◦ with anproduct onion-like and 5-fluoro-2-(1-fluorovinyl)-4 odor. When a chloroformH-1,3-dithiin solution of(1712) aswas the minor heated product. in a sealed If a mixture tube at of 80 12 Cand for 4 h it decomposeda five-fold excess giving of a ethyl mixture acrylate of 5-fluoro-3-(1-fluorovinyl)-3,4-dihydro-1,2-dithiin was heated in a sealed tube at 100 °C for 6 h, a 70:30 mixture (16) as of the the major productregioisomeric and 5-fluoro-2-(1-fluorovinyl)-4 Diels-Alder adducts (19H and-1,3-dithiin 20) of ethyl (17) acrylate as the minor and 2-fluorothioacrolein product. If a mixture (15) ofwas12 and a five-foldobtained excess in 60% of ethylyield, acrylatein accord waswith heated the prop inosed a sealed mechanism tube at for 100 intramolecular◦C for 6 h, aconversion 70:30 mixture of of the regioisomericallicin to thioacrolein Diels-Alder and 2-propenesulfenic adducts (19 and 20acid) of (Scheme ethyl acrylate 1) [1,2]. andIt is 2-fluorothioacroleinassumed that the second (15) was obtainedproduct in 60% in decomposition yield, in accord of 12 with, 2-fluoro-2-propenesulfenic the proposed mechanism acid for (14 intramolecular), self-condenses conversion back to 12. of allicin Treatment of 9 with cysteine in NH4OH afforded fluorodeoxyalliin (13; S-2-fluoro-2-propenyl-L- to thioacrolein and 2-propenesulfenic acid (Scheme1)[ 1,2]. It is assumed that the second product in cysteine) in 73% yield. After crystallization 13 could be characterized by X-ray crystallography as the 12 14 12 decompositionpure enantiomer of ,shown 2-fluoro-2-propenesulfenic in Figure 1. Structural data acid is given ( ), self-condensesin Table 1. Two molecules back to co-crystallized. Treatmentin the structure; of 9 theywith are cysteine just slightly in NH different4OHafforded in bond dimensions. fluorodeoxyalliin Oxidation (13 of; anS-2-fluoro-2-propenyl- aqueous solution L-cysteine)of 13 (30% in 73%H2O2) yield. gave fluoroalliin After crystallization (22; S-2-fluoro-2-propenyl-13 could beL characterized-cysteine S-oxide; by 82% X-ray yield). crystallography When 22 as thewas pure treated enantiomer with powdered shown fresh in garlic Figure, the1. presence Structural of small data amounts is given of in 12 Table and isomers1. Two of molecules mono- co-crystallizedfluoroallicin in 23 the along structure; with major they amounts are just of slightly allicin 4 differentwas suggested in bond by DART-MS dimensions. analysis; Oxidation the of 19 an aqueouspresence solution of 12 and of 2313 was(30% also H consistent2O2) gave with fluoroalliin the results (22 from; S-2-fluoro-2-propenyl- F-NMR spectroscopy.L -cysteine S-oxide; 82% yield).When When a concentrated22 was treated solution with powderedof 12 in 1,1,1,3,3,3-hexafluoro-2 fresh garlic, the presence-propanol of small was amountskept at room of 12 and isomerstemperature of mono-fluoroallicin for 16 h it was converted23 along withto a 1:1 major mixture amounts of 12 and of trifluoroajoene allicin 4 was suggested18 ((E,Z)-1-(2-fluoro- by DART-MS 3-((2-fluoroallyl)sulfinyl)prop-1-en-1-yl)-2-(2-fluoroallyl)disulfane) in 59% yield, based on converted analysis; the presence of 12 and 23 was also consistent with the results from 19F-NMR spectroscopy. When a concentrated solution of 12 in 1,1,1,3,3,3-hexafluoro-2-propanol was kept at room temperature for 16 h it was converted to a 1:1 mixture of 12 and trifluoroajoene 18 ((E,Z)-1- (2-fluoro-3-((2-fluoroallyl)sulfinyl)prop-1-en-1-yl)-2-(2-fluoroallyl)disulfane) in 59% yield, based on Molecules 2017, 22, 2081 4 of 20

Molecules 2017, 22, 2081 4 of 20 converted 12. The choice of 1,1,1,3,3,3-hexafluoro-2-propanol follows from the observation that partial conversion12. The ofchoice12 to of a mixture1,1,1,3,3,3-hexafluoro-2-propanol of 16–18 occurred in isopropanol, follows andfrom the the belief observation that the higher that aciditypartial of Molecules 2017, 22, 2081 4 of 20 1,1,1,3,3,3-hexafluoro-2-propanolconversion of 12 to a mixture of would16–18 occurred favor formation in isopropanol, of 18. When and the the belief same that reaction the higher was conducted acidity of 1,1,1,3,3,3-hexafluoro-2-propanol◦ would favor formation of 18. When the same reaction was at 6012C,. The a mixture choiceof of12 1,1,1,3,3,3-hexafluoro-2-propanol, 16 and 18 was formed, suggesting follows that from heat the favors observation formation that of 16partial. While 18 conducted at 60 °C, a mixture of 12, 16 and 18 was formed, suggesting that heat favors formation of couldconversion be characterized of 12 to a as mixture a mixture of 16 with–18 occurred12 by NMR in isopropanol, and liquid and chromatography-high the belief that the higher resolution acidity mass 16. While 18 could be characterized as a mixture with 12 by NMR and liquid chromatography-high spectrometry,of 1,1,1,3,3,3-hexafluoro-2-propanol separation of 12 and 18 waswould not favor possible formation with the of chromatographic18. When the same methods reaction employed, was resolution mass spectrometry, separation of 12 and 18 was not possible with the chromatographic unfortunatelyconducted precludingat 60 °C, a mixture biological of 12 studies, 16 and of18 18was. formed, suggesting that heat favors formation of methods employed, unfortunately precluding biological studies of 18. Treatment16. While 18 ofcould disulfide be characterized11 with as liquefied a mixture elementalwith 12 by NMR sulfur and at liquid its melting chromatography-high point of 120 ◦C Treatment of disulfide 11 with liquefied elemental sulfur at its melting point of 120 °C for 1 h, as for 1resolution h, as described mass spectrometry, previously forseparation4 [23], affordedof 12 and polysulfane18 was not possible mixture with21 thewith chromatographic 4–15 sulfur atoms, described previously for 4 [23], afforded polysulfane mixture 21 with 4–15 sulfur atoms, with withmethods compounds employed, containing unfortunately 4–6 sulfur precluding atoms biological predominating. studies of 18 A. proposed mechanism, initiated compoundsTreatment containing of disulfide 4–6 sulfur 11 with atoms liquefied predominating. elemental sulfur A proposed at its melting mechanism, point of 120 initiated °C for 1by h, [2,3]- as by [2,3]-sigmatropic rearrangement of disulfide 11 (Scheme3), is consistent with computational studies sigmatropicdescribed rearrangementpreviously for 4of [23], disulfide afforded 11 polysulfane(Scheme 3), mixture is consistent 21 with with 4–15 computational sulfur atoms, studieswith showingshowingcompounds that that fluorine containingfluorine substitution substitution4–6 sulfur on atoms the on 2-position predominating.the 2-posi oftion the A allylof proposed the group allyl mechanism, has group little effecthas initiated onlittle rearrangement byeffect [2,3]- on to rearrangement11asigmatropic[24]. Analysis rearrangementto 11a of the[24]. complex Analysis of disulfide polysulfane of the 11 complex (Scheme mixture polysulfane3), wasis consistent performed mixture with by was computational ultra-performance performed studiesby ultra- liquid + chromatographyperformanceshowing that liquid (UPLC) fluorine chromatography silver substitution coordination on(UPLC) the ion 2-posi spraysilvertion masscoordination of spectrometrythe allyl ion group spray ((Ag has )CIS-MS)mass little spectrometry effect (Figure on 2) as 19 well((Agrearrangement as+)CIS-MS) by F NMR (Figure to and11a 2) DART-MS[24]. as wellAnalysis as techniques,by of 19 Fthe NMR complex asand described DART-MSpolysulfane more techniques, mixture fully was in as the performed described experimental bymore ultra- fully section andin elsewheretheperformance experimental [23 liquid]. section chromatography and elsewhere (UPLC) [23]. silver coordination ion spray mass spectrometry ((Ag+)CIS-MS) (Figure 2) as well as by 19F NMR and DART-MS techniques, as described more fully in the experimental section and elsewhere [23].

Figure 1. Molecular structure of the L-enantiomer of fluorodeoxyalliin (13, S-2-fluoro-2-propenyl-L- Figure 1. Molecular structure of the L-enantiomer of fluorodeoxyalliin (13, S-2-fluoro-2-propenyl- cysteine);Figure 1.non-hydrogen Molecular structure atoms ofar ethe represented L-enantiomer by thermalof fluorodeoxyalliin ellipsoids at ( 13the, S 50%-2-fluoro-2-propenyl- probability level.L- L-cysteine);cysteine); non-hydrogen non-hydrogen atomsatoms ar aree represented represented by by thermal thermal ellipsoid ellipsoidss at the at 50% the 50%probability probability level. level.

Scheme 3. Conversion of disulfide 11 to polysulfane mixture 21 through condensation with molten SchemeScheme 3. 3.Conversion Conversion of of disulfide disulfide 1111 toto polysulfanepolysulfane mixture mixture 2121 throughthrough condensation condensation with with molten molten sulfur.sulfur. Several Several equivalents equivalents of of SS8 8 cancan react,react, losinglosing different numbersnumbers of of sulfur sulfur atom atom groupings, groupings, sulfur. Several equivalents of S8 can react, losing different numbers of sulfur atom groupings, followed followedfollowed0 by by SN S2N′ 2reaction′ reaction with with 2-fluoroallyl 2-fluoroallyl sulfoniusulfonium species giving giving polysulfane polysulfane mixture mixture 21 21. . by SN2 reaction with 2-fluoroallyl sulfonium species giving polysulfane mixture 21.

Molecules 2017, 22, 2081 5 of 20 Molecules 2017, 22, 2081 5 of 20

+ FigureFigure 2. 2. UPLCUPLC (Ag (Ag+)-CIS-MS)-CIS-MS selective selective ion ion chromatogram chromatogram of polysulfane of polysulfane mixture mixture 21, 21with, with 4 to 4 15 to 107 15 sulfur atoms: C6H8F2107S4- Ag complex, m/z 352.7, 6.78/6.85 min (split peak due to column issues); sulfur atoms: C6H8F2S4- Ag complex, m/z 352.7, 6.78/6.85 min (split peak due to column issues); 107 107 C6H8F2S5- Ag complex, m/z 384.7, 7.71 min; C6H8F2S6- Ag complex, m/z 416.7, 8.41 min; C6H8F2S5-107Ag complex, m/z 384.7, 7.71 min; C6H8F2S6-107Ag complex, m/z 416.7, 8.41 min; C6H8F2S7- 107 107 C6H8F2S7- Ag complex, m/z 448.7, 9.00 min; C6H8F2S8- Ag complex, m/z 480.7, 9.50 min; 107Ag complex, m/z 448.7, 9.00 min; C6H8F2S8-107Ag complex, m/z 480.7, 9.50 min; C6H8F2S9-107Ag 107 107 C6H8F2S9- Ag complex, m/z 512.6, 9.97 min; C6H8F2S10- Ag complex, m/z 544.5, 10.38 min; complex, m/z 512.6, 9.97 min; C6H8F2S10-107Ag complex, m/z 544.5, 10.38 min; C6H8F2S11-107Ag complex, 107 107 C6H8F2S11- Ag complex, m/z 576.4, 10.7 min; and C6H8F2S12- Ag complex, m/z 608.4, 11.1 min. m/z 576.4, 10.7 min; and C6H8F2S12-107Ag complex, m/z 608.4, 11.1 min. With additional signal With additional signal amplification, the following low abundance complexes could also be seen: amplification, the following low abundance complexes could also be seen: C6H8F2S13-107Ag complex, 107 107 C6H8F2S13- Ag complex, m/z 640.3, 11.43 min; C6H8F2S14- Ag complex, m/z 672.3, 11.73 min; 6 8 2 14 107 6 8 2 15 107 m/z 640.3, 11.43107 min; C H F S - Ag complex, m/z 672.3, 11.73 min; C H F S - Ag complex, m/z C6H8F2S15- Ag complex, m/z 704.2, 12.05 min. The insert shows molecular ions m/z 352.73 and 704.2, 12.05 min. The insert shows molecular107 ions m/z 352.73109 and 354.73 corresponding to C6H8F2S4- 107354.73 corresponding109 to C6H8F2S4- Ag and C6H8F2S4- Ag.109 The chromatogram selecting for Ag and C1096H8F2S4- Ag. The chromatogram selecting for C6H8F2Sn- Ag complexes was very similar. C6H8F2Sn- Ag complexes was very similar. All new compounds were fully characterized by spectroscopic methods, either as pure compounds,All new or compounds in the case were of 18 fully mixed characterized with 12, 19 bymixed spectroscopic with 20, and methods, 21, as mixtures. either aspure While compounds, synthesis ofor influorine-substituted the case of 18 mixed analogs with of12 , garlic-der19 mixedived with organosulfur20, and 21, ascompounds mixtures. Whilein Scheme synthesis 1 is of straightforward,fluorine-substituted their analogs accessibility of garlic-derived is limited by organosulfur the high cost compounds of starting in material Scheme1 9 is. While straightforward, 12 and 18 aretheir chiral accessibility due to the is limitedsulfinyl by group, the high thiosulfinates cost of starting such material as 12 are9 .stereochemically While 12 and 18 arelabile chiral [1], dueso no to effortthe sulfinyl was made group, to synthesize thiosulfinates enantiomerically such as 12 are pure stereochemically material. labile [1], so no effort was made to synthesize enantiomerically pure material.

Molecules 2017, 22, 2081 6 of 20

Table 1. Selected bond distances (Å) and angles (deg.) in 13. Atom labeling corresponds to that in Figure1.

Bond Lengths S(1)-C(10) 1.806(3) C(10)-C(11) 1.472(5) F(1)-C(5) 1.360(4) S(1)-C(9) 1.812(3) C(9)-C(8) 1.512(5) C(1)-C(2) 1.524(4) O(5)-C(7) 1.242(4) C(11)-C(12) 1.287(5) C(5)-C(6) 1.286(5) O(4)-C(7) 1.238(4) S(2)-C(4) 1.803(4) C(5)-C(4) 1.477(5) N(2)-C(8) 1.479(4) S(2)-C(3) 1.815(3) C(2)-N(1) 1.488(4) F(2)-C(11) 1.362(4) O(1)-C(1) 1.242(4) C(2)-C(3) 1.510(4) C(7)-C(8) 1.525(4) O(2)-C(1) 1.247(4) Angles C(4)-S(2)-C(3) 100.4(2) C(6)-C(5)-F(1) 119.4(3) N(1)-C(2)-C(1) 109.6(3) O(1)-C(1)-O(2) 125.8(3) C(6)-C(5)-C(4) 129.0(4) C(3)-C(2)-C(1) 112.4(3) O(1)-C(1)-C(2) 118.5(3) F(1)-C(5)-C(4) 111.5(3) C(2)-C(3)-S(2) 113.4(2) O(2)-C(1)-C(2) 115.7(3) N(1)-C(2)-C(3) 110.8(3) C(5)-C(4)-S(2) 113.4(3)

2.2. Biological Studies of Fluorinated Analogues of Garlic-Derived Organosulfur Compounds Since ancient times, garlic has been used worldwide as a folk medicine for prevention of coronary thrombosis, stroke, and atherosclerosis, and for treatment of infections and diseases including vascular disorders, by reducing serum cholesterol levels and increasing blood coagulation times, and for treatment of cancer, by inhibiting proliferation of cancer cells [1–8]. Here, we will compare the anti-angiogenesis and antithrombotic activity of certain fluorinated analogues of garlic-derived organosulfur compounds with their non-fluorinated counterparts.

2.2.1. Anti-Angiogenesis Efficacy of Fluorinated Compounds in the Chick Chorioallantoic Membrane (CAM) Model Angiogenesis, the formation of new blood vessels from pre-existing capillaries and circulating endothelial precursors, plays a key role in tumor growth and metastasis as well as other physiological and pathological processes such as embryonic development, chronic inflammation, and wound healing. It is regulated by factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), tumor necrosis factor-α (TNF-α), and interleukin-8 (IL-8) [25]. We found that bFGF-mediated angiogenesis as examined with the CAM model was significantly inhibited (p < 0.001) by allicin (1), ajoene (8), difluoroallicin 12 or S-2-fluoro-2-propenyl-L-cysteine (13), with greater inhibition (p < 0.1) by 12 as compared to the other organosulfur compounds tested (Figure3). Difluoroallicin 12 was significantly (p < 0.01) more potent than allicin (1) or S-2-fluoro-2-propenyl-L-cysteine (13) in inhibiting angiogenesis-mediated by bFGF. Furthermore, bis(2-fluoroallyl)polysulfane mixture 21 demonstrated comparable anti-angiogenesis efficacy against FGF-mediated angiogenesis in the CAM model to that of ajoene (Figure4). Representative illustrations of the anti-angiogenesis efficacy of the various organosulfur compounds against FGF are shown in Figure5, which shows the relative qualitative images that were quantitated for n = 6–8 CAM per arm. Additionally, a similar pattern of anti-angiogenesis efficacy for the various organosulfur compounds was also demonstrated against VEGF in the CAM model (Figure6). At the end of the CAM study the membrane was dissected for quantitative imaging and the effect on survival of embryos was documented. Significantly, none of the treatment arms showed any mortality of the embryos at the doses tested. To complement the above CAM model, the dorsal skinfold chamber technique [26] would be a very desirable method for future use in our anti-angiogenesis studies. Molecules 2017, 22, 2081 7 of 20

FGF+Ajoene(8) FGF+Allicin(1) FGF+Difluoroallicin FGF+S-2-fluoro-2- (12) propenyl-L-cysteine(13)

Figure 3. Inhibition of bFGF-mediated angiogenesis in the CAM model by 4 µg/20 µL/CAM of ajoene (8), allicin (1), difluoroalliicin 12, and S-2-fluoro-2-propenyl-L-cysteine (13); * p < 0.01 difluoroallicin (12) versus allicin (1) and ** p < 0.001 for all versus control FGF. Data showed that all organosulfur compounds tested demonstrated significant anti-angiogenesis activity, with greater efficacy for difluoroallicin 12 versus allicin (1). Data represent Mean (% inhibition of angiogenesis) ± SEM, n = 6–8 CAM/group.

FGF+Ajoene(8) Fluoroallyl)polysufane(21)

Figure 4. Anti-angiogenesis efficacy of ajoene compared with 1,2-bis(2-fluoroallyl)polysulfane mixture (21) against FGF-mediated angiogenesis in the CAM [***p < 0.001] by 4 µg/20 µL/CAM of ajoene (8) or 1,2-bis(2-fluoroallyl)polysulfane (21). Data showed significant (***p < 0.001) and comparable anti-angiogenesis efficacy between ajoene (8) and 1,2-bis (2-fluoroallyl) polysulfane (21). Data represent Mean (% inhibition of angiogenesis) ± SEM, n = 8 CAM/group. Molecules 2017, 22, 2081 8 of 20

PBS (Vehicle) FGF (1 μg) FGF + Ajoene (8; 4 μg)

FGF + Allicin (1; 4 μg) FGF + Difluoroallicin (12; 4 μg)

Figure 5. Representative images of CAM neovascularizationneovascularization induced by bFGF and inhibited by the various garlic-derived compounds compounds including including ajoene ajoene (8 (8),), allicin allicin (1 ()1 )and and difluoroalliicin difluoroalliicin (12 (12), ),each each at at4 μ4g/20µg/20 μL/CAM.µL/CAM. The The images images shown shown are are representative representative sing singlele images images selected selected for for illustration illustration of of the general anti-angiogenesis efficacy efficacy of the organosulfur compounds and not for quantitative purposes.

Figure 6. RepresentativeRepresentative images images of of CAM CAM neovasculari neovascularizationzation induced induced by by VEGF and inhibited by garlic-derived compounds, including including ajoene ajoene ( (88),), allicin allicin ( (11),), difluoroalliicin difluoroalliicin 1212,, and and SS-2-fluoro-2--2-fluoro-2- propenyl-L-cysteine-cysteine ( (1313),), each at 44 µμg/20g/20 μµL/CAM. The The images images shown shown are are representative single images selectedselected for for illustration illustration of the of general the general anti-angiogenesis anti-angiogenesis efficacy ofefficacy the organosulfur of the organosulfur compounds compoundsand not for quantitativeand not for quantitative purposes. purposes.

Molecules 2017, 22, 2081 9 of 20

2.2.2. Anti-Angiogenesis Efficacy of Organosulfur Compounds in the Mouse Matrigel® Model Difluoroallicin 12 was tested at different concentrations in the Matrigel® plug assay for its anti-angiogenesis activity. Inhibition of angiogenesis was assessed by measuring the hemoglobin level in the Matrigel® plug after 14 days of implant. Difluoroallicin 12 demonstrated a dose-dependent inhibition of angiogenesis with a maximal inhibition at 10–30 µg/plug (Figure7).

Difluoroallicin

Figure 7. Dose-response inhibition of FGF-induced angiogenesis (hemoglobin, mg/dL) in FGF enriched Matrigel® with or without different doses ranging from 1–100 µg of difluoroalliicin (12) in mice. Data represent Mean Angiogenesis index (Hemoglobin, mg/dL) ± SEM, n = 18 Matrigel® implant/group.

2.2.3. Effect of Fluorinated and Normal Organosulfur Compounds on Platelet Aggregation and Platelet/Fibrin Clot Dynamics Antithrombotic drugs are classified as either anticoagulants or antiplatelet drugs. Anticoagulants slow down clotting, thereby reducing fibrin formation and preventing clots from forming and growing, while antiplatelet agents prevent from aggregating (clumping), also preventing clots from forming and growing. The effect of various organosulfur compounds on platelet aggregation was assessed using human whole blood impedance aggregometry-induced by collagen. Allicin (1; 62 µM) and difluoroallicin 12 (51 µM) demonstrated the most potent inhibition of platelet aggregation (p < 0.001) as compared to ajoene or S-2-fluoro-2-propenyl-L-cysteine (13) (Figure8). At these concentrations, allicin was slightly more effective compared to difluoroallicin. For allicin, we determined platelet ATP secretion in response to collagen in human whole blood, demonstrating similar antiplatelet activity in inhibiting platelet activation by suppressing platelet ATP secretion and aggregation (Figure9). Mayeux et al. tested the effects of allicin on platelet aggregation and showed dose dependency up to 100 µM. They also show data for cAMP levels over this concentration range [27]. Our concentrations of 10 µg/mL, equivalent to 62 µM, are comparable to the range used by Mayeux. It would be desirable to compare the activity of difluoroallicin 12 and the other fluorosulfur compounds in future studies of the type shown in Figure9. The effect of the various organosulfur compounds on clot strength was assessed using human whole blood thrombelastography (TEG). Difluoroallicin 12 showed the most potent inhibition of clot strength at 10–30 µg (p < 0.001) as compared to ajoene (8), allicin (1) or S-2-fluoro-2-propenyl-L-cysteine (13) (Figure 10). The other organosulfur compounds showed very modest effects on clot dynamics (Figure 10). Figure 11 shows representative TEG tracings of clot dynamics and kinetics, with the Molecules 2017, 22, 2081 10 of 20 Molecules 2017, 22, 2081 10 of 20 compared to other organosulfur compounds tested. This is in agreement with previous reports on the antiplatelet effects of allicin [22]. Distinctly greater inhibition of platelet/fibrin clot dynamics and most potent inhibition by difluoroallicin 12 compared to a more modest effect by allicin (1). The degree kinetics,of inhibition which of platelettranslate aggregation into greater for difluoroallicinantithrombotic12 efficacy,and allicin was (1) isobserved greater compared for difluoroallicin to other comparedorganosulfur to other compounds organosulfur tested. compounds This is in agreement tested. Difluoroallacin with previous reports gave a on concentration-dependent the antiplatelet effects inhibitionof allicin [of22 ].clot Distinctly strength greater with inhibition maximal of inhibition platelet/fibrin at 20–30 clot dynamics μg/mL and(Figure kinetics, 11). which We, translateand other investigatorsinto greater [21,22], antithrombotic reported efficacy, inhibition was observedof platelet for aggregation difluoroallicin by comparedallicin. None to other of the organosulfur organosulfur compoundscompounds tested tested. showed Difluoroallacin any effects gave on aendothelial concentration-dependent cell (HUVEC) inhibitionviability over of clot 24 strengthh. with maximal inhibition at 20–30 µg/mL (Figure 11). We, and other investigators [21,22], reported inhibition of platelet aggregation by allicin. None of the organosulfur compounds tested showed any effects on endothelial cell (HUVEC) viability over 24 h.

Control Ajoene(8) Allicin(1) Difluoroallicin S-2-Fluoro-2-propenyl-L- (12) cysteine(13) Figure 8. Effect of various garlic-derived compounds including ajoene (8), allicin (1), difluoroalliicin (12), andFigure S-2-fluoro-2-propenyl- 8. Effect of various garlic-derivedL-cysteine (13 compounds) on human including platelet ajoene aggregation (8), allicin at (1 ),10 difluoroalliicin μg/mL. Data (12 represent), meanand plateletS-2-fluoro-2-propenyl- aggregation ±L standard-cysteine (deviation,13) on human n = platelet3–4. aggregation at 10 µg/mL. Data represent mean platelet aggregation ± standard deviation, n = 3–4.

FigureFigure 9.9. RepresentativeRepresentative platelet platelet aggregation aggregation and ATP secretionsecretion inin response response to to collagen collagen (A ()A and) and collagencollagen plus plus allicin allicin at at 1 1 and and 10 10 μµg/mL ( (BB).). The The representative illustration illustration showed showed inhibition inhibition of of both both plateletplatelet aggregation aggregation and and ATP ATP secretion versusversus controlcontrol (collagen). (collagen).

Molecules 2017, 22, 2081 11 of 20

Molecules 2017, 22, 2081 11 of 20 Molecules 2017, 22, 2081 11 of 20

Figure 10. Effect of various garlic-derived compounds including ajoene (8), allicin (1), difluoroalliicin Figure(12), Figureand 10. Effect 10.S-2-fluoro-2-propenyl- Effect of various of various garlic-derived garlic-derivedL-cysteine compounds compounds (13) on including includingclot strength ajoene ajoene (8 ( ),8),in allicin allicin human ( 1(1),),difluoroalliicin difluoroalliicin blood using (12 ), andthrombelastographyS(-2-fluoro-2-propenyl-12), and S-2-fluoro-2-propenyl- at differentL-cysteine amount.L (-cysteine13) Data on clot repr(13 strength)esent on meanclot in human strengthclot strength blood in using human(MA, thrombelastography mm)blood ± standardusing atdeviation, differentthrombelastography n amount.= 4. Data at represent different amount. mean clot Data strength represent (MA, mean mm) clot± strengthstandard (MA, deviation, mm) ± standard n = 4. deviation, n = 4.

Figure 11. Representative tracing of clot kinetic for control human blood and blood treated with either Figureallicin 11. 11. Representative(1Representative) or difluoroalliicin tracing tracing 12 atof 20–30 clot of clot kineticμg. The kinetic foclotr controlstrength for control human is measured human blood by and bloodthe widthblood and of treated bloodthe tracing with treated at either the with eitherallicinend ( allicin1) orof difluoroalliicintracing. (1) or For difluoroalliicin the 12positive at 20–30 control, 12μg.at The there 20–30 clot is strengthµa g.large The width is clotmeasured seen, strength which by the is means measuredwidth that of theeffective by tracing the blood width at the of theend tracingofcoagulation tracing. at theFor occurred. endthe ofpositive Difluoroallicin tracing. control, For 12 thethere showed positive is adistinct large control, andwidth gr thereeater seen, inhibition is which a large meansat width20 μg/mL that seen, concentrationseffective which blood means thatcoagulation effectivecompared occurred. blood to allicin. coagulation Difluoroallicin This can be occurred. seen 12 inshowed the Difluoroallicingreen distinct trace andrelative gr12eater toshowed the inhibition control distinct black at 20 tracing. and μg/mL greater concentrations inhibition at 20comparedµg/mL to concentrations allicin. This can compared be seen in to the allicin. green Thistrace canrelative be seen to the in control the green black trace tracing. relative to the control black tracing.

Molecules 2017, 22, 2081 12 of 20

While comparable antiplatelet activity is shown for allicin (1) and difluoroallicin 12 (Figure8), greater anticoagulant activity (inhibition of fibrin and clot strength) is shown for 12 compared to 1 (Figures 10 and 11). This is the first report illustrating the impact of allicin on coagulation activation-mediated clot kinetics. The dose-response relationship for the different compounds tested showed a slope with different degrees of inhibition of clot strength (MA, mm), suggesting a biological effect in a concentration-dependent manner on the fibrin kinetics, versus all or none, which would imply lack of toxicity (Figures 10 and 11). These data suggest an effective anti-clotting efficacy in human whole blood. Figure 10 indicates that difluoroallicin 12 at 10 µg/mL is in fact somewhat more effective in inhibiting clot strength than allicin (1) and the other compounds tested. While experiments on the effects of our new fluorinated organosulfur compounds on cells in culture would be worthwhile, based on the report that different cell lines vary in their sensitivity to allicin [28], it would not necessarily be easy to extrapolate results with cells in culture to platelets in human blood.

2.2.4. Effect of Fluorine Substitution on Biological Activity The premise in this work was that fluorine substitution would enhance the electrophilic character of allicin and other garlic-derived organosulfur compounds, which in turn would enhance their reactivity toward biological thiol groups, considered to be the defining feature of their biological activity [13–15]. While the modest, but significant, enhanced antithrombotic potency of difluoroallicin 12 compared to allicin (1) is in line with our expectations, the enhanced potency may also reflect changes in lipophilicity and membrane fluidity [29] associated with fluorine substitution. The anti-angiogenesis activity of 1,2-bis(2-fluoroallyl)polysulfane (21) is notable. Because 21 is a mixture, it would be worthwhile examining the biological activity of individual 1,2-bis(2-fluoroallyl)polysulfanes, such as the disulfide (11) and tetrasulfide (21, n = 4), among others.

3. Materials and Methods

3.1. General Procedures

NMR spectra were recorded in CDCl3, unless otherwise indicated, on a 400 Ultrashield, spectrometer (Bruker, Billerica, MA, USA) operating at 400 MHz for proton, 100 MHz for carbon, and or a Bruker 600 Avance III HD with QCI Cryoprobe operating at 600 MHz for proton, 125 MHz for carbon and 376.5 MHz for 19F. The chemical shifts (δ) are indicated in ppm downfield from tetramethylsilane, with the internal standard being the residual CHCl3 (δ 7.27 ppm for proton and 19 δ 77.23 for carbon spectra). F-NMR chemical shifts were relative to CFCl3 at δ 0.00 ppm. Infrared spectra of the neat compounds were recorded on a UATR 2 FTIR (Perkin Elmer, Boston, MA USA). GC-MS were obtained using the Hewlett Packard 6890 GC and a 5972A selective mass detector (Agilent Technologies, Inc., Santa Clara, CA, USA). A DART-AccuTOF (JEOL USA, Inc., Peabody, MA, USA) time-of-flight mass spectrometer operating in positive or negative ion mode was employed with a polyethylene glycol spectrum as reference standard for exact mass measurements (PEG average molecular weight: 600). The atmospheric pressure interface was typically operated at the following potentials: orifice 1 = 15 V; orifice 2 = 5 V; ring lens = 3 V. The RF ion guide voltage was generally set to 800 V to allow detection of ions greater than m/z 80. The DART ion source was operated with helium gas at 200 ◦C, and a grid voltage = 530. 3-Chloro-2-fluoroprop-1-ene (9) was obtained from Synquest Laboratories (Alachua, FL, USA). Selected 1H-, 13C- and 19F-NMR and DART-MS data for new compounds are available in the Supporting Materials.

3.2. Synthesis of Fluorinated Garlic Organosulfur Compounds S-(2-Fluoroallyl)ethanethioate (10). Potassium thioacetate (6.58 g, 57.6 mmol, 1.1 equiv.) was added to a solution of 3-chloro-2-fluoroprop-1-ene (9; 5 g, 53.2 mmol) in THF (150 mL). The mixture was stirred overnight to give an orange-yellow solution with a white precipitate. The solution was filtered through a silica gel pad and then concentrated in vacuo to yield the title compound (10; 3.23 g, 23.4 mmol, 44%) Molecules 2017, 22, 2081 13 of 20

1 3 2 as a yellow liquid; H-NMR (600 MHz, CDCl3) δ 4.53 (dd, JHF = 43.9 Hz, JHH = 3.06 Hz, CF=CHH, 3 2 3 1H), 4.48 (dd, JHF = 75.9 Hz, JHH = 3.06 Hz, CF=CHH, 1H), 3.58 (d, JHF = 17.3 Hz, CH2, 2H), 2.20 13 1 2 (s, CH3, 3H); C- NMR (100 MHz, CDCl3) δ 193.7 (s), 161.5 (d, JCF = 257.0 Hz), 92.6 (d, JCF = 19 Hz), 2 19 30.2 (s), 29.6 (d, JCF = 31.3 Hz); F-NMR (376.5 MHz, CDCl3) δ −98.3 to −98.2 (m); IR: νmax 2981, 1697, 1673, 1275, 1132, 1105, 944, 857, 846, 624 cm−1; HRMS (ESI) exact mass calculated for [M + H]+ (C5H8OFS) requires m/z 135.0279, found m/z 135.0290. 1,2-Bis(2-fluoroallyl)disulfane (11). Sodium chips (0.267 g, 11.63 mmol, 1.07 equiv.) were added to anhydrous methanol (100 mL) at 0 ◦C. The mixture was stirred until the sodium disappeared completely. S-(2-Fluoro-2-propenyl)ethanethioate (10; 1.5 g, 10.87 mmol) was added at 0 ◦C followed by iodine (1.407 g, 11.09 mmol, 1.02 equiv.) after 2 h. The mixture was stirred for 30 min and then concentrated in vacuo and purified by silica gel column chromatography () to give the title compound (11; 0.46 g, 1 3 2 2.53 mmol, 23%) as a yellow liquid; H-NMR (600 MHz, CDCl3) δ 4.66 (dd, JHF = 96.5 Hz, JHH = 3 Hz, 3 2 3 CF=CHH, 1H), 4.63 (dd, JHF = 107 Hz, JHH = 3 Hz, CF=CHH, 1H), 3.42 (d, JHF = 18.9 Hz, CH2, 2H); 13 1 2 2 C-NMR (100 MHz, CDCl3) δ 161.6 (d, JCF = 252.0 Hz), 94.1 (d, JCF = 19.0 Hz), 39.9 (d, JCF = 30.2 Hz); 19 F-NMR (376.5 MHz, CDCl3) δ −100.57 to −100.30 (m, 2F); IR: νmax 2919, 1668, 1271, 1133, 946, 891, −1 + 855, 841, 732 cm ; HRMS (ESI) exact mass calculated for [M + H] (C6H9F2S2) requires m/z 183.0114, found m/z 183.0124. In other runs, higher yields (38%) were obtained. Difluoroallicin (12; S-(2-Fluoroallyl) 2-fluoroprop-2-ene-1-sulfinothioate). A 32% peracetic acid/acetic acid solution (0.58 g, 2.6 mmol, 1.04 equiv.) was added dropwise to a solution of bis-(2-fluoro-2-propenyl) disulfide (11; 0.46 g, 2.5 mmol) in chloroform (50 mL) at 0 ◦C. The reaction mixture was vigorously ◦ stirred at 0 C for 45 min as anhydrous Na2CO3 (4.0 g, 37.7 mmol, 15.1 equiv.) was added in small portions. The mixture was stirred for an additional 1 h at 0 ◦C and then filtered through a pad of Celite ◦ and anhydrous MgSO4. The filtrate was concentrated in vacuo at 0 C to yield the crude, water-soluble title compound 12, which was purified by preparative TLC (4:1 :diethyl ether) to give pure 12 1 as yellow liquid with an onion-like odor (0.21 g, 1.06 mmol, 42%); H-NMR (600 MHz, CDCl3) δ 4.90 3 2 3 2 (dd, JHF = 136 Hz, JHH = 3.30 Hz, S(O)–CH2–CF=CHH, 1H), 4.85 (dd, JHF = 201 Hz, JHH = 3.60 Hz, 3 2 S(O)–CH2–CF=CHH, 1H), 4.75 (dd, JHF = 69.15 Hz, JHH = 3.3 Hz, S–CH2–CF=CHH, 1H), 4.71 3 2 4 2 (dd, JHF = 97.8 Hz, JHH = 3.60 Hz, S–CH2–CF=CHH, 1H), 4.00 (dd, JHF = 35 Hz, JHH = 19.25 Hz, 4 2 4 S(O)–CHH, 1H), 3.99 (dd, JHF = 21 Hz, JHH = 8.4 Hz, S(O)–CHH, 1H), 3.86 (dd, JHF = 73.5 Hz, 2 4 2 13 JHH = 17.25 Hz, S–CHH, 1H), 3.87 (dd, JHF = 42.0 Hz, JHH = 17.1 Hz, S–CHH, 1H); C-NMR 2 2 2 (100 MHz, CDCl3) δ 32.5 (d, JCF = 31.0 Hz) 58.8 (d, JCF = 29.0 Hz), 94.1 (d, JCF = 16.9 Hz), 2 1 1 19 98.3 (d, JCF = 17.9 Hz) , 155.4 (d, JCF = 259 Hz) 160.5 (d, JCF = 161 Hz); F-NMR (376.5 MHz, CDCl3) δ −99.65 to −99.44 (m, 1F), −94.90 to −94.62 (m, 1F); IR: νmax 2917, 1669, 1407, 1272, 1081 −1 + (S=O), 945 cm ; HRMS (ESI) exact mass calculated for [M + H] (C6H9OF2S2) requires m/z 199.0063, found m/z 199.0080. Storage at −40 ◦C or lower is recommended to avoid decomposition. Vacuum distillation was not attempted due to the presumed thermal sensitivity of 12.

S-2-Fluoro-2-propenyl-L-cysteine (13; 2-Amino-3-((2-fluoroallyl)thio)propanoic acid). 3-Chloro-2-fluoroprop- 1-ene (9) (0.94 g, 10 mmol) was added to an ice-cold solution of L-cysteine (1.21 g, 10 mmol) in ammonium hydroxide (40 mL) with vigorous stirring. The mixture was stirred at 0 ◦C for 60 min and filtered; the filtrate was concentrated in vacuo (<40 ◦C) to a small volume and filtered. The solid was washed repeatedly with ethanol, dried in vacuo, and recrystallized from 2:3 water: ethanol to yield 13 ◦ 1 as white needles (1.31 g, 7.31 mmol, 73% yield), mp 220–223 C; H-NMR (D2O): δ 4.75–4.51 (m, 2H), 19 3.90–3.87 (m, 1H), 3.32 (d, 2H, J = 16 Hz), 3.13–2.99 (m, 2H); F-NMR (D2O): δ −101.30 to −101.58 13 (m, 1F); C-NMR (CCl3): δ 172.41, 161.73, 159.21, 53.27, 31.63–31.35 (d), 31.16. Single crystals were analyzed by X-ray crystallography as described in Section 3.3 below to provide the crystal structure of the L-enantiomer shown in Figure1. Trifluoroajoene (18) [(E,Z)-1-(2-Fluoro-3-((2-fluoroallyl)sulfinyl)prop-1-en-1-yl)-2-(2-fluoroallyl)disulfane]. A solution of 12 (200 mg; 1.01 mmol) was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (0.50 mL) Molecules 2017, 22, 2081 14 of 20 at room temperature. The mixture was immediately analyzed by DART-MS which showed the formation of trace amounts of trifluoroajoene 18. The mixture was stirred at room temperature for 16 h whereupon analysis by DART-MS showed a 1:1 mixture of F-allicin and F-ajoene, with no F-dithiin present. The mixture was concentrated in vacuo to yield a faintly yellow liquid (180 mg). 1H-, 13C- and 19F-NMR spectroscopy indicated a 1:1 mixture of difluoroallicin 12 and trifluoroajoene 18, with no difluorodithiins present, corresponding to a 59% yield of 18 based on 12 converted. A small sample was stirred for 32 h but no further conversion of 12 to 18 was observed. Attempts to purify 18 by silica gel chromatography or HPLC were unsuccessful, precluding biological testing; 1H-NMR (400 MHz, 3 13 CDCl3) 18: δ 5.91 (d, 2H, JHF = 120.9), 5.90 (s, 1H), 4.4 (m, 2H), 3.8 (m, 6H); 12 as above; C-NMR (100 MHz, CDCl3) 18: δ 59.75 (d, 1C, J = 27.70 Hz), 60.02 (d, 1C, J = 27.59 Hz), 68.82 (d, 1C, J = 33.33 Hz), 69.32 (s, 1C), 69.81 (d, 1C, J = 33.23 Hz), 109.44 (d, 1C, J = 36.08 Hz), 108.57 (d, 1C, J = 283.20 Hz), 110.86 19 (d, 1C, J = 247.17 Hz) , 121.75 (d, 1C, J = 278.34 Hz); 12 as above; F-NMR (376.5 MHz, CDCl3) 18: δ −78 (s, 1F), −128 (s,1F), −140 (s, 1F); 12 as above; HRMS (ESI) exact mass calculated for [M + H]+ (C9H12F3OS3) requires m/z 289.0002, found m/z 288.9992. 5-Fluoro-3-(1-fluorovinyl)-3,4-dihydro-1,2-dithiin (16). S-(2-Fluoroallyl) 2-fluoroprop-2-ene-1-sulfinothioate (12; 50.0 mg) in CHCl3 (1 mL) was placed in a glass tube which was sealed under vacuum and heated at 80 ◦C for 4.0 h. The reaction mixture was analyzed by GC-MS and the major dithiin isomer 16 1 was purified by flash chromatography (n-pentane); H-NMR (400 MHz, CDCl3) δ 3.61–2.84 (m, 2H), 13 3.93–3.99 (m, 1H), 4.44–4.57(m, 1H), 4.39–4.94 (m, 1H), 6.09–6.13 (m, 1H). C-NMR (100 MHz, CDCl3) δ 30.3 (dd, J = 28.0, 5.0 Hz), 45.5 (dd, J = 30.0, 11.5 Hz), 93.3 (d, J = 19.5 Hz), 101.0 (d, J = 27.0 Hz) 157.0 19 (d, J = 395.0 Hz) 160.0 (d, J = 395.0 Hz); F-NMR (376.5 MHz CDCl3): δ −99.80 to −99.60 (m, 1F), −1 −88.83 to −88.78 (m, 1F). IR: νmax 2853, 1668, 1420, 1257, 1192, 1100, 928 cm ; DART-HR-TOF-MS + [M + H] C6H7F2S2 found m/z 180.9958, requires m/z 180.9957. 2-Fluoroacrolein-ethyl acrylate Diels-Alder adducts: Ethyl 5-fluoro-3,4-dihydro-2H-thiopyran-2-carboxylate (19) and ethyl 5-fluoro-3,4-dihydro-2H-thiopyran-3-carboxylate (20). A mixture of difluoroallicin 12 (98.0 mg, 0.5 mmol) and ethyl acrylate (250.0 mg, 2.5 mmol) in CHCl3 (2.5 mL) were placed in a glass tube. The tube was sealed under vacuum and heated at 100 ◦C for 6.0 h. The reaction mixture was analyzed by NMR and the crude material was purified by using flash chromatography (n-pentane/Et2O), affording 70:30 (19:20) mixture (56.0 mg, 60% yield). It was not possible to separate completely 19 (major) from 20 (minor) by flash chromatography. DART-HR-TOF-MS (M + H) C8H12FO2S found m/z 1 191.0538, requires m/z 191.0542. 19 (Major 70%): H-NMR (400 MHz, CDCl3) δ 5.67 (d, J = 17.7 Hz, 1H), 4.15–4.22 (m, 2H, overlap with 20), 3.05–3.08 (m, 1H), 2.85–2.98 (m, 2H overlap with 20), 2.49–2.63 13 (m, 2H, overlap with 20), 1.26–1.30 (m, 3H, overlap with 20); C-NMR (100 MHz, CDCl3: δ 172.0, 153.5 (d, J = 259.7 Hz), 97.3 (d, J = 27.5 Hz), 61.2, 40.7 (m, overlap with 20), 27.2 (m, overlap with 20), 19 26.4, 14.0 (overlap with 20); F-NMR (376.5 MHz CDCl3): δ −96.26 to −96.21 (m). 20 (Minor 30%): 1 H-NMR (400 MHz, CDCl3) δ 5.61 (d, J = 17.7 Hz, 1H), 4.15–4.22 (m, 2H, overlap with 19), 3.35–3.43 (m, 1H), 2.85–2.98 (m, 2H), 2.49–2.63 (m, 2H, overlap with 19), 1.26–1.30 (m, 3H, overlap with 19); 13 C-NMR (100 MHz, CDCl3) δ 170.4, 151.4 (d, J = 259.7 Hz), 95.7 (d, J = 27.5 Hz), 61.6, 40.6 (m, overlap 19 with 19), 27.2 (m, overlap with 19), 23.4, 14.0 (overlap with 19); F- NMR (376.5 MHz CDCl3): δ −96.09 to −96.04 (m). Bis(2-fluoroallyl) polysulfane mixture (21). A 10 mL round-bottomed flask containing sublimed sulfur ◦ (S8, 0.256 g, 1.0 mmol) was placed in an oil bath pre-heated to 120 C. When the sulfur had completely melted to a clear, straw-colored liquid, disulfide 11 (0.182 g, 1.0 mmol) was added all at once to the magnetically stirred liquid. After 30 min, the initial two-layer liquid mixture became a clear, homogeneous solution. Samples were withdrawn from the reaction mixture for analysis at various time points, e.g., 30 min, 1 h, 2 h, 4 h and 5 h. The withdrawn samples were dissolved in CDCl3 permitting both NMR and reversed phase HPLC analysis to be performed on the same sample. For the 2 h sample, disulfide 11 eluted at 2.65 min, S8 eluted between the heptasulfide and octasulfide at 22.2 min, and the highest polysulfane seen, eluting at 159 min, had a chain of 26 sulfur atoms. Molecules 2017, 22, 2081 15 of 20

By UPLC-(Ag+)-CIS-MS, chains containing up to 15 sulfur atoms could be characterized as their 107Ag and 109Ag adducts (see supporting information and discussion elsewhere [23]). 1H-NMR spectroscopic analysis after 30 min shows the CH2 proton signals as doublets centered at δ 3.39 [area 0.35, S2], 3.52 [area 0.08, S3], 3.61 [area 0.08, S4], 3.63 [area 0.16, S5], 3.64 [area 0.50, ≥S6]; after 5 h 1H-NMR spectroscopic analysis shows 3.39 [area 0.06 S2], 3.52 [area 0.08 S3], and 3.64 19 [area 0.95, ≥S6]. After 1.25 h, F-NMR (376.5 MHz CDCl3) showed a series of multiplets centered at δ −100.3 (11;C6H8F2S2) and −100.0 (C6H8F2S3) down to −99.5 ppm (C6H8F2S>5). DART was used to obtain HRMS (ESI) exact mass data on C6H9F2Sn components in a mixture examined after 1.25 h, of heating. The lighter components gave better exact mass agreement compared to the + heavier components: calculated for [M + H] (11;C6H9F2S2) requires m/z 183.0114, found m/z + 183.0117; calculated for [M + H] (C6H9F2S3) requires m/z 214.9835, found m/z 214.9835; calculated for + + [M + H] (C6H9F2S4) requires m/z 246.9555, found m/z 246.9563; calculated for [M + H] (C6H9F2S5) + requires m/z 278.9276, found m/z 278.9242; calculated for [M + H] (C6H9F2S6) requires m/z 310.8997, + found m/z 310.8985; calculated for [M + H] (C6H9F2S7) requires m/z 342.8717, found m/z 342.8808; + + calculated for [M + H] (C6H9F2S8) requires m/z 374.8438, found m/z 374.9639; calculated for [M + H] + (C6H9F2S9) requires m/z 406.8159, found m/z 406.9403; calculated for [M + H] (C6H9F2S10) requires m/z 438.7880, found m/z 438.9084. UPLC-(Ag+)-CIS-MS analysis of (21) is listed in Table S2.

(+)-S-2-Fluoro-2-propenyl-L-cysteine S-oxide (22). S-2-Fluoro-2-propenyl-L-cysteine (13) (1.18 g, 6.59 mmol) was added to 12 mL of water with vigorous stirring until it dissolved completely. Hydrogen peroxide (30%, 2.56 g) was added to the solution which was then stirred for 45 min at RT. The water was removed under vacuum and the title compound 22 (1.04 g, 82%, 5.33 mmol) was obtained as a white ◦ 1 powder, mp 168–171 C; H-NMR (D2O) 5.03 (dd, 2H, J = 3 Hz), 4.86–4.82 (m, 2H) 4.08–3.75 (m, 2H), 19 3.50-3.26 (m, 2H); F-NMR (D2O) −94.84 to −95.12 (m, 1F), −95.18 to −95.47 (m, 1F).

3.3. X-ray Crystallographic Structural Determination for 13 The single crystal diffraction data for 13 was measured on a SMART APEX CCD X-ray diffractometer (Bruker, Madison, WI, USA) equipped with a graphite monochromated Mo Kα radiation source (λ = 0.71073 Å) at T = 100(2) K. Data reduction and integration were performed with the Bruker software package SAINT (Version 8.38A). Data were corrected for absorption effects using the empirical methods as implemented in SADABS. Both SAINT and SADABS are part of Bruker APEX3 software package (Version 2016.9-0): Bruker AXS, 2016. The structure was solved by SHELXT (Version 2014/5) [30] and refined by full-matrix least-squares procedures using the Bruker SHELXTL (Version 2016/6) XL refinement program version 2016/6 software package [30]. All non-hydrogen atoms (including those in disordered parts) were refined anisotropically. The H-atoms were also included at calculated positions and refined as riders, with Uiso(H) = 1.2 Ueq(C) and Uiso(H) = 1.5 Ueq(N) for groups. Crystallographic data, details of the data collection and structure refinement for all reported compounds are listed in Table S1.

3.4. Chick Chorioallantoic Membrane (CAM) Protocols

3.4.1. Growth Factor-Mediated Angiogenesis

Chick Chorioallantoic Membrane (CAM) Model of Angiogenesis Neovascularization was examined in the CAM model, as previously described [31–33]. Briefly, ten-day old chick embryos were purchased from Spafas, Inc. (Preston, CT, USA) and incubated at 37 ◦C with 55% relative humidity. Two small holes were created in the shell at the air sac and on the broadside of the egg using a hypodermic needle directly over an avascular portion of the embryonic membrane identified by candling. A false air sac beneath the second hole was created by the application of negative pressure at the first hole causing the CAM to separate from the shell. A window, approximately 1.0 cm2, was cut in the shell over the dropped CAM with a small crafts Molecules 2017, 22, 2081 16 of 20 grinding wheel (Dremel, Division of Emerson Electric Co., Racine, WI, USA), allowing direct access to the underlying CAM. FGF2 (1 µg/mL) or VEGF (2 µg/mL) were used as a standard pro-angiogenic agent to induce new blood vessel branches added into the CAM membrane of the 10-day old embryos. Sterile disks of #1 filter paper (Sigma Aldrich, St. Louis, MO, USA) were pre-treated with 3 mg/mL of cortisone acetate and air dried under sterile conditions. FGF-2 and VEGF were dissolved in PBS (phosphate buffered saline). FGF2 (1 µg /mL) or VEGF (2 µg /mL) treated filter disks were placed in avascular area of the membrane. The test compound was added to membrane at different concentrations using fixed 20 µL volume.

Microscopic Analysis of CAM Sections After incubation at 37 ◦C with 55% relative humidity for 3 days, the CAM tissue directly beneath each filter disk was resected from control and treated CAM samples. Tissues were washed 3 times with PBS, placed in 35-mm Petri dishes (Nalge Nunc, Rochester, NY, USA) and examined under an SV6 stereomicroscope (Karl Zeiss, Thornwood, NY, USA) at 50× magnification. Digital images of CAM sections exposed to filters were collected, using a 3-CCD color video camera system (Toshiba America, New York, NY, USA), and analyzed with Image-Pro software (Media Cybernetics, Silver Spring, MD, USA). The numbers of vessel branch points contained in a circular region equal to the area of each filter disk counted. One image was counted in each CAM preparation, and findings from 6–8 CAM preparations/group were analyzed for each treatment condition.

3.4.2. Mouse Matrigel®-Growth Factors Implant Angiogenesis Model The model is as previously described [33], and was performed in accordance with institutional guidelines for animal safety and welfare. Female mice C56/BL aged 5–6 weeks and body weights of 20 g were purchased from Taconic Farms (Hudson, NY, USA). Animals were maintained under specific pathogen-free conditions and housed four animals per cage, under controlled conditions of temperature (20–24 ◦C) and humidity (60%–70%) and a 12 h light/dark cycle. Water and food were provided ad libitum. The in vivo study was carried out in the animal facility of the Veterans Affairs (VA) Medical Center, Albany, NY, USA and the experimental protocol was approved by the VAIACUC. Mice were acclimatized for 5 days prior to the start of experiments. Matrigel® Matrix High Concentration with growth factors to promote the angiogenesis was used and the mix was injected four times subcutaneously at 100 µL/animal. Animals in the control group were injected just with 100-µL volumes of Matrigel®. The fluorinated and non-fluorinated organosulfur compound were used at three different doses (3, 10 or 30 µg /10 µL). Animals in the treatment groups were injected with 3, 10 or 30 µg of compounds into the 100 µL Matrigel® and cell mix. All groups had three mice per group, with a total of 12 Matrigel® subcutaneous injection per group. At day 18 post plug implant, all animals were sacrificed and hemoglobin contents were quantitated using spectrophotometry.

3.4.3. Determination of Hemoglobin (Hb) Levels (Measure of Angiogenesis Index) Matrigel® plug Hb content was indexed as a measure of new vascularity. Matrigel® plugs were placed into a 0.5 mL tube containing double distilled water and then homogenized for 5–10 min. The samples were centrifuged at 5000× g for 10 min and then the supernatants were collected. A volume of 50 µL of supernatant was mixed with 50 µL of Drabkin’s reagent and the mixture was kept at room temperature for 15–30 min, at which time 100 µL was placed in a 96-well plate and absorbance measured at 540 nm with a Microplate Manager ELISA reader (BioRad Laboratories, Hercules, CA, USA). Hb concentration is expressed as mg/dL based on comparison with a standard curve.

3.4.4. Platelet Aggregation in Whole Blood (Impedance Method) Blood was obtained from healthy human volunteers (n = 6) who were drug-free for a minimum of 2 weeks before the blood sample collection. Blood was collected in Vacutainer tubes containing Molecules 2017, 22, 2081 17 of 20

(3.2% citrate); 500 µL of blood incubated with 393 µL saline, 5 µL test compounds or 5 µL saline and then 2 µL of the platelet agonist collagen (1–5 µg) was added to induce platelet aggregation and ATP secretion. An electrical impedance aggregation measurement was performed on the whole-blood aggregometer, which is equipped with automated calibration and readout functions. An aliquot of whole blood (0.5 mL) was diluted with an equivalent volume of isotonic saline and incubated for 5 min at 37 ◦C. The impedance of each sample was monitored in sequential 1 min intervals until a stable baseline was established. After a stable baseline established, collagen and aggregation was monitored for 10 min, and the final increase in ohms over this period was displayed [34].

3.4.5. Global Coagulation Assay (Thrombelastography)

Blood Sampling Siliconized Vacutainer tubes (Becton Dickinson, Rutherford, NJ, USA) were used to collect whole blood. To maintain a ratio of citrate to whole blood of 1:9 (v/v), the tubes contained 3.2% trisodium citrate. Blood samples were placed on a slow speed rocker until TEG analysis.

Thrombelastography (TEG) A Whole Blood Coagulation Analyzer, Model 5000 Thrombelastograph (Haemoscope Corporation, Skokie, IL, USA) was used. TEG is based on the measurement of the physical viscoelastic characteristics of blood clots. An oscillating plastic cylindrical cuvette (“cup”) and a coaxially suspended stationary piston (“pin”) with a 1-mm clearance between the surfaces are used to monitor clot formation at 37 ◦C. Every 4.5 s, with a 1-s mid-cycle stationary period, the cup oscillates in either direction, resulting in a frequency of 0.1 Hz. A torsion wire that acts as a torque transducer suspends the pin. Fibrin fibrils link the cup to the pin during clot formation, and the rotation of the cup is transmitted to the pin via the viscoelasticity of the clot. Customized software (Haemoscope Corporation) and an IBM-compatible personal computer display the rotation. The pin’s torque is plotted as a function of time, as shown by the different TEG clot parameters [35–38].

4. Conclusions The organosulfur compounds tested in the CAM model have anti-angiogenesis activity against either FGF or VEGF that might have impact on pathological angiogenesis-mediated disorders such as cancer and macular degeneration, among others. Additionally, difluoroallicin 12 demonstrated effective anti-platelet activity comparable to allicin (1), and anticoagulant activities greater than allicin and the other organosulfur compounds tested. This is the first report illustrating the impact of allicin and its homologs on the inhibition of coagulation activation-mediated clot kinetics. The absence of embryo mortality in all treatment arms of the CAM study suggests the lack of toxicity for 1, 8, 12 and 13 at the doses tested. Furthermore, the dose-response relationship for 1, 8, 12, and 13 shows a slope with different degrees of inhibition of clot strength, suggesting a concentration-dependent biological effect on the fibrin kinetics, which is also inconsistent with toxicity of these compounds. The enhanced antithrombotic potency of 12 compared to 1 is in line with expectations based on enhanced electrophilic character and/or changes in lipophilicity and membrane permeability due to fluorine substitution. These anti-thrombotic activities could have significant impact on cardiovascular and inflammatory diseases, which are associated with increased thrombosis risk. Further studies on these and related compounds are warranted pending enhanced availability of precursors such as 3-chloro-2-fluoroprop-1-ene or related compounds [39]. While we encourage others to explore this area, it should be noted that 3-chloro-2-fluoroprop-1-ene and, particularly, 2-fluoro-2-propenol are toxic (LD50 for skin penetration, 200 and 4 mg/kg, respectively) and therefore should be handled with great care [40–42]. Molecules 2017, 22, 2081 18 of 20

X-Ray Crystallographic Data: CCDC1577315 contains the supplementary crystallographic data, which can be obtained free of charge from Cambridge Crystallographic Data Centre, www.ccdc.cam.ac.uk/data_request/cif. Supplementary Materials: Supplementary Materials are available online. Documentation for the X-ray structure (Table S1), UPLC-(Ag+)-CIS-MS analysis of bis(2-fluoro-2-propenyl) polysulfanes (21) (Table S2), and selected 1H-, 13C- and 19F-NMR and DART-MS data for new compounds are available online. Acknowledgments: We acknowledge support from the National Science Foundation (CHE-0744578; CHE-1265679; CHE-1337594 [MRI]; CHE-1429329 [MRI]) and the University at Albany (E.B.) and the Pharmaceutical Research Institute for funding the biological studies (S.M.), and thank Alexander Filatov and Zheng Wei for performing the X-ray structural analysis and Robert Sheridan for UPLC-MS studies. We dedicate this paper with our warmest wishes to Professor E.J. Corey on his 90th birthday. Author Contributions: E.B. and S.A.M. conceived and designed the experiments; B.B., S.G., A.V., K.W., S.S.M., K.G. and M.Y. performed the experiments and analyzed the data; E.B. and S.A.M. wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds described in the text are not available from the authors.

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